Endothelial cells lining the interior of blood vessels hinder transport of drug carriers from the bloodstream into diseased tissues. In order to overcome this obstacle, drug carriers can be targeted to cell-surface receptors that mediate transcytosis across this barrier. However, the mechanism by which drug carriers detach from the barrier after transport and migrate through the underlying extracellular matrix (ECM) is still unknown. Therefore, the overall goal of this work is to examine these aspects for better understanding of the mechanisms involved in regulating and modulating endothelial barrier function. In doing so, this work will guide translational drug delivery for treatment of diseases where transendothelial transport is required. As a clinically-relevant example of an application, this proposal will explore these questions in the case of polymer nanocarriers targeted to intercellular adhesion molecule-1 (ICAM-1) and carrying an enzyme cargo (acid sphingomyelinase, ASM) for treatment of type A-B Niemann-Pick Disease, a lysosomal storage disorder caused by deficiency of lysosomal ASM, which results in severe pulmonary and brain dysfunction. Specifically, ICAM-1 is an interesting and relevant target to study the mechanism of drug carrier transport because it is highly expressed on the endothelium during pathological conditions, and facilitates transport of natural (leukocytes) and artificial (ICAM-1-targeted drug carriers) ligands across endothelial barriers. In agreement with literature showing the interplay between ICAM-1 and matrix metalloproteinases (MMPs), particularly MMP-9 and MMP-2, preliminary results indicate that carrier binding to ICAM-1 causes MMP-9 and MMP-2 secretion from endothelial cells, and MMP-9 contributes to ICAM-1 cleavage. Therefore, it is hypothesized that MMP release upon carrier binding to ICAM-1 results in (a) ICAM-1 cleavage, allowing carrier detachment from the barrier after transcytosis, and (b) ECM degradation, allowing carrier penetration into tissues. To investigate these hypotheses, this proposal examines the contribution of MMP-9 and MMP-2 in (1) ICAM-1 cleavage, (2) carrier transport across endothelial barriers, and (3) ECM degradation for carrier penetration in both cell culture and animal models. First, ELISA and Western blotting will be used to assess MMP secretion and ICAM-1 cleavage, as well as examine the correlation between MMP-mediated release of ICAM-1 and carrier biodistribution. Second, radioisotope tracing will be used to examine carrier transport across endothelial barriers via ICAM-1 cleavage. Third, ECM degradation and carrier penetration will be assessed using gelatin zymography, fluorescence microscopy, immunohistochemistry, and TEM imaging. In each case, cell culture and animal models deficient in MMP-9 and/or MMP-2 will be used to identify their role in these processes. Through completion of these aims, this work stands to improve a number of drug therapies requiring transport across impermeable endothelial barriers. This research will also significantly contribute to training the PI as an independent investigator, thereby enabling future research for the advancement of biomedical technologies.